CN109136353A - A kind of method that low sequence relies on high-order constant temperature exponential amplification detection microRNA - Google Patents
A kind of method that low sequence relies on high-order constant temperature exponential amplification detection microRNA Download PDFInfo
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Abstract
The invention discloses a kind of methods that low sequence relies on high-order constant temperature exponential amplification detection microRNA, this method is specifically able to detect the template T1 and T2 of target microRNA by design, template T1 and T2, archaeal dna polymerase, nicking restriction endonuclease, dNTP are mixed, and it is reacted under the conditions of 35~50 DEG C, detect amplification curve, determine whether contain target microRNA in sample according to amplification curve and standard curve, the method for the present invention can further calculate out the concrete content of target microRNA in sample.The method of the present invention amplification efficiency is high, and high sensitivity shortens analysis time；This method is small to the sequence dependent of target microRNA, versatile；It is wide to detect the range of linearity, specificity is good；Amplification system is simple, can develop into kit and to marketing.
Description
Technical field
The invention belongs to nucleic acid quantification detection fields, and in particular to a kind of low sequence dependence high-order constant temperature exponential amplification detection
The method of microRNA (hereinafter referred to as miRNA).
Background technique
MiRNA is a kind of endogenic non-coding microRNA, is about 18-24 nucleotide, it has gene expression
Regulating and controlling effect plays an active part in cell Growth and Differentiation and apoptosis activity in vivo.A large amount of result of study shows miRNA's
It is closely related between expression and cancer or certain diseases.Therefore, deep reason is not only facilitated to the quantitative detection of miRNA
Its mechanism of action is solved, and Clinics and Practices to disease and the exploitation of related gene drug etc. are of great significance.But by
It is short and small in the sequence of miRNA, the sequence similarity with height, with differing only by 1-2 base between miRNAs and in practical sample
Content is very low in product, and accurately quantitative detection miRNA is a bigger challenge.
Identify and the common method of quantitative miRNA has Northern engram technology, Microarray method and reverse transcription fixed
Measure the side PCR (Reverse Transcription-Quantitative Polymerase Chain Reaction, RT-qPCR)
Method.Northern engram technology is the standard method based on hybridization means detection miRNA, and this method needs a large amount of sample and divides
It is cumbersome, time-consuming from enriching step, and can not the delicately subtle miRNAs of distinguishing sequence difference.The side Microarray
Method can carry out high-throughput miRNA analysis, while detect to a variety of miRNAs, have unique advantage, but the spy of method
The opposite sex is poor, sensitivity is lower, and the production and detection of micro-array chip are expensive.RT-qPCR is to detect miRNA very
Sensitive method, the sample size needed is few, but since miRNA sequence is short, could expand after needing reverse transcription, while this method needs
It is accurately controlled the temperature of each amplification step.
In order to preferably meet the requirement of the clinical miRNA of detection immediately, the constant temperature for not needing thermal cycle and alternating temperature process expands
Increase detection technique to have gradually developed.Rolling circle amplification (RCA), ring mediated isothermal amplification (LAMP), exponential amplification (EXPAR) etc. are all
It is representative constant-temperature amplification detection technique.Wherein, rolling circle amplification and loop-mediated isothermal amplification technique are when detecting miRNA
Long, the about 1.5h with good specificity, but the time required to these technologies.It was set up by D.J.Galas etc. in 2003
Constant temperature exponential amplification reaction (Isothermal Exponential Amplification Reaction) will test the time contracting
It is as short as 30min, promises to be the technology for realizing detection (Point-of-care testing, POCT) immediately.But existing constant temperature
There is apparent sequence dependent in exponential amplification reaction, can table for the amplification template of different target molecule miRNA design
Reveal different amplification efficiencies.2012, Niemz team passed through the template that Computer Design constant temperature exponential amplification reacts, and just sent out
Under present identical thermodynamic condition, different templates shows different amplification capabilities, the amplification of constant temperature exponential amplification reaction
Efficiency relies on template sequence to a certain extent.Therefore, constant temperature exponential amplification is improved, develop it is a kind of more rapidly sensitive and
The low amplification technique of sequence dependent is of great significance.
Summary of the invention
In order to avoid the strong problem of conventional thermostatic exponential amplification methods sequence dependent, while amplification efficiency is improved, shortened
Analysis time realizes that the purpose quickly analyzed, the present invention devise a kind of high-order constant temperature exponential amplification techniques.High-order constant temperature index
The amplification efficiency of amplification technique mainly determines by template, is influenced by target microRNA sequence small, breaches current constant temperature and refers to
Number amplification method relies on sequence strong limitation, can be applied to detect a variety of short-chain nucleic acids, while having amplification rate fast, clever
The advantages that sensitivity height, high specificity.
The purpose of the present invention is to provide a kind of low sequence rely on high-order constant temperature exponential amplification detection microRNA template and
Method.
The technical solution used in the present invention is:
A kind of low sequence relies on the template of high-order constant temperature exponential amplification detection microRNA, and the template is by template T1 and T2
Composition, template T1 and T2 are DNA sequence dna；
5 ' the terminal sequences of template T1 and T2 are identical, contain two sections of identical sequence x, contain between two sections of sequence x
The specific site of nicking restriction endonuclease identification, is denoted as x- specific site-x；
3 ' the terminal sequences of template T2 can be with the 5 ' terminal sequence reverse complementals of target microRNA；
3 ' the terminal sequences of template T1 can be located at upper with the intermediate sequence reverse complemental of template T2, the intermediate sequence of the T2
It states between the 3 ' terminal sequences of T2 and the 5 ' terminal sequences of T2；
The intermediate sequence of template T1 can be with the 3 ' terminal sequence reverse complementals of target microRNA, the intermediate sequence of the T1
Between the 3 ' terminal sequences of above-mentioned T1 and the 5 ' terminal sequences of T1；
In the presence of T1, T2 and target microRNA three are common, two two parts reverse complemental of sequence between three, jointly
Hybridization forms T-type structure；And under archaeal dna polymerase effect, the 3 ' ends of target microRNA can be expanded with T1 template, be expanded
Increase the sequence with 5 ' terminal sequence reverse complemental of T1 out, is denoted as x '-specific site-x '；3 ' the ends of T1 can be with T2 template simultaneously
It is expanded, the sequence with 5 ' terminal sequence reverse complemental of T2 amplified is denoted as x '-specific site-x '；
Above-mentioned x '-specific site-x ' and x- specific site-x is reverse complementary sequence, and the two combines the double-strand formed can
It is cut by nicking restriction endonuclease, the site of nicking restriction endonuclease cutting is located in x '-specific site-x ' chain.
Preferably, the 3 ' terminal sequences of the template T2 can be with the 5 ' terminal sequence reverse complementals of target microRNA, the section
5 ' terminal sequence the length of target microRNA account for the 8/21~12/21 of target microRNA total length.
Preferably, 3 ' terminal sequence reverse complementals of the intermediate sequence of the template T1 and target microRNA, this section of target
3 ' terminal sequence the length of microRNA account for the 8/21~12/21 of target microRNA total length.
Preferably, the length of the sequence x is 14~25nt.
Preferably, the 3 ' terminal sequences of the template T1 can be with the intermediate sequence reverse complemental of template T2, this section of reverse mutual
The sequence length of benefit is 8~12bp.
Preferably, the nicking restriction endonuclease is selected from one of Nt.BstNBI, Nt.BsmAI, Nt.AlwI.
Preferably, when object to be measured microRNA is let-7b, the template is TCC ACC GTT TAC TTC
TAA TGA CTC TCC ACC GTT TAC TTC TAA CCA CAA CCC CAA AAA and TCC ACC GTT TAC TTC
TAA TGA CTC TTC CAC CGT TTA CTT CTT TTT TGG GAC CAA CCT ACT ACC TCA TT；
When object to be measured microRNA is miR-374a, the template is TCC ACC GTT TAC TTC TAA
TGA CTC TCC ACC GTT TAC TTC TAA TTA CAA CCC CAAAAA and TCC ACC GTT TAC TTC TAA
TGACTC TTC CAC CGT TTA CTT CTT TTT TGG GAC ATA CAA TCT GAT AAG TT；
When object to be measured microRNA is miR-762, the template is TCC ACC GTT TAC TTC TAA TGA
CTC TCC ACC GTT TAC TTC TGC TCG GCA CCC CAA AAA and TCC ACC GTT TAC TTC TAA TGA
CTC TTC CAC CGT TTA CTT CTT TTT TGG GAC CCC GGC CCC AGC CCC TT。
A kind of method that low sequence relies on high-order constant temperature exponential amplification detection microRNA, comprising the following steps: will be to be measured
Sample, the template T1 and T2 described in any of the above embodiments for being able to detect target microRNA, archaeal dna polymerase, nicking restriction endonuclease,
DNTP mixing, entire reaction system is reacted under the conditions of 35~50 DEG C, detects amplification curve, bent according to amplification curve and standard
Line determines whether contain target microRNA in sample；And the specific of target microRNA in sample can be further calculated out and contained
Amount.
Preferably, archaeal dna polymerase 0.04~0.10U/ of concentration μ L, nicking inscribe enzyme concentration 0.20 in the reaction system~
0.50U/ μ L, 0.005~0.200 μM of template T2 concentration.
Most preferably, archaeal dna polymerase concentration 0.06U/ μ L in the reaction system, nicking inscribe enzyme concentration 0.40U/ μ L,
0.075 μM of template T2 concentration.
Preferably, the concentration of template T1 is 0.08~0.12 μM in the reaction system.
Preferably, DNA polymerase buffer liquid, fluorescent dye, nicking inscribe enzyme buffer liquid are also contained in the reaction system.
Preferably, the standard curve is vertical sit with the POI value of amplification curve using target microRNA concentration as abscissa
Mark.
The beneficial effects of the present invention are:
The method of the present invention designs two exponential amplification templates by improving to widely applied exponential amplification methods,
Partial complementarity, common hybridization form T-type structure to sequence two-by-two between target microRNA three；Then recycle this improved
Template carries out the concentration of exponential amplification detection target to be measured.The invention has the following advantages that
1) amplification efficiency is high, and high sensitivity shortens analysis time；
2) this method is small to the sequence dependent of target miRNA, versatile；
3) the detection range of linearity is wide, and specificity is good；
4) amplification system is simple, can develop into kit and to marketing.
Detailed description of the invention
The low sequence of Fig. 1 present invention relies on the schematic diagram of high-order constant temperature exponential amplification detection microRNA, Target in figure: mesh
Mark miRNA, extension: amplification, polymerase:DNA polymerase, nicking: cutting, nicking enzyme: nicking
Restriction endonuclease, release: release.
The other amplification curve diagram of Fig. 2 difference group；
Fig. 3 modified constant temperature exponential amplification of the present invention and traditional constant temperature exponential amplification curve comparison figure；
Amplification curve diagram of Fig. 4 the method for the present invention to each member of let-7 family；
Amplification curve diagram when Fig. 5 let-7b is mixed with other miRNA of let-7 family；
Fig. 6 various concentration miR-374a and let-7b POI histogram；
Fig. 7 various concentration miR-762 and let-7b POI histogram；
The fluorescent amplification curve of Fig. 8 detection various concentration let-7b；
The standard curve of Fig. 9 detection miRNA.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
A kind of low sequence of embodiment 1 relies on the template of high-order constant temperature exponential amplification detection microRNA
As shown in Figure 1, designing amplification the template T1 and T2 of two partial complementarities according to target miRNA；Template T1's and T2
5 ' terminal sequences are identical, and containing two sections of identical sequence x, (length is 14~25nt), contains nicking between two sections of sequence x
The specific site of restriction endonuclease identification, is denoted as x- specific site-x；
3 ' the terminal sequences of template T2 can (5 ' terminal sequence length accounts for target with the 5 ' terminal sequences of target microRNA
The 8/21~12/21 of microRNA total length) reverse complemental；
3 ' the terminal sequences of template T1 can be with the intermediate sequence reverse complemental (length is 8~12bp) of template T2, the T2
Intermediate sequence be located between the 3 ' terminal sequences of above-mentioned T2 and the 5 ' terminal sequences of T2；
The intermediate sequence of template T1 can (3 ' terminal sequence length accounts for target with the 3 ' terminal sequences of target microRNA
The 8/21~12/21 of microRNA total length) reverse complemental, the intermediate sequence of the T1 is located at the 3 ' terminal sequences and T1 of above-mentioned T1
5 ' terminal sequences between；
In the presence of T1, T2 and target microRNA three are common, two two parts reverse complemental of sequence between three, jointly
Hybridization forms T-type structure；And two-way amplification, i.e., the 3 ' of target microRNA are carried out simultaneously along T1, T2 under archaeal dna polymerase effect
End can be expanded with T1 template, amplified the sequence with 5 ' terminal sequence reverse complemental of T1, be denoted as x '-specific site-x '；
3 ' the ends of T1 can be expanded simultaneously with T2 template, amplify the sequence with 5 ' terminal sequence reverse complemental of T2, be denoted as x '-spy
Ectopic sites-x '；
X '-specific site-x ' segment and x- specific site-x segment are reverse complementary sequence, x '-specific site-x ' segment
In containing nicking restriction endonuclease identification specific site, can by nicking restriction endonuclease (selected from Nt.BstNBI, Nt.BsmAI,
One of Nt.AlwI) cutting；Through inscribe enzyme effect, generating the short-chain nucleic acids x', x' complementary with x sequence can continue and swim
From amplification template T1 and T2 in x sequence continue hybridization expanded, it is seen that high-order constant temperature exponential amplification reaction amplification effect
Rate is close to 2 × 2n, to improve sensitivity, reduce sequence dependent.
The method that a kind of low sequence of embodiment 2 relies on high-order constant temperature exponential amplification detection microRNA
To detect in let-7 family for let-7b, according to the sequence for the target let-7b to be detected, part therewith is designed
Complementary template T1 and template T2, particular sequence is as shown in table 1.
The sequence of 1 let-7b of table and its template T1 and template T2
A and B reaction solution is prepared on ice, and A reaction solution is divided into tetra- kinds of A1, A2, A3, A4:
A1 includes inscribe enzyme buffer liquid, template T1, template T2, dNTP, target let-7b；
A2 includes inscribe enzyme buffer liquid, template T1, dNTP, target let-7b；
A3 includes inscribe enzyme buffer liquid, template T2, dNTP, target let-7b；
A4 includes inscribe enzyme buffer liquid, template T1, template T2, dNTP.
B reaction solution includes DNA polymerase buffer solution, Vent (exo-) archaeal dna polymerase, restriction endonuclease Nt.BstNBI, SYBR
Green I fluorescent dye.
By A1 and B, A2 and B, A3 and B, A4 and B carried out after mixing respectively at 45 DEG C real-time exponential amplification reactions (mixing
0.1 μM of T1 final concentration in system, 0.075 μM of T2 final concentration, restriction endonuclease final concentration 0.4U/ μ L, the final concentration 0.06U/ μ of polymerase
L), with BIO-RAD CFX ConnectTMQuantitative fluorescent PCR monitors amplification curve,
Amplification curve compares POI value as shown in Fig. 2, amplification efficiency in the presence of let-7b, T1 and T2 three are common is maximum
(point of inflection values, the greatest gradient of real-time fluorescence intensity curve corresponding time), let-7b, T1 and
The T-type structure that T2 three is formed makes proliferation time shorten 30min or more, and only need to expand 10min can be obtained testing result, and
Other groups, which are both needed to amplification 40min or more, just result.
The method that a kind of low sequence of embodiment 3 relies on high-order constant temperature exponential amplification detection microRNA
To detect in let-7 family for let-7b, according to the sequence for the target let-7b to be detected, part therewith is designed
Complementary template T1 and template T2, particular sequence is as shown in table 1.
A the and B reaction solution of high-order constant temperature exponential amplification techniques (TEXPAR) of the present invention is prepared on ice, and A reaction solution includes
Inscribe enzyme buffer liquid, template T1, template T2, dNTP, target let-7b；B reaction solution includes polymerization enzyme buffer solution, Vent
(exo-) polymerase, restriction endonuclease Nt.BstNBI, SYBR Green I fluorescent dye.By A and B mixing, (T1 is whole in mixed system
0.1 μM of concentration, 0.075 μM of T2 final concentration, restriction endonuclease final concentration 0.4U/ μ L, the final concentration 0.06U/ μ L of polymerase), then exist
50 DEG C carry out real-time exponential amplification reaction, with BIO-RAD CFX ConnectTMQuantitative fluorescent PCR monitors amplification curve.
Comparative example 1
To detect in let-7 family for let-7b, according to the sequence for the target let-7b to be detected, traditional constant temperature is designed
Exponential amplification template T:5'-AAC CAC ACA ACC TAC TAC CTC AAA CAG ACT CAA ACC ACA CAA CCT
ACT ACC TCA A-3'(SEQ ID NO:4).
Reaction solution, is made into C and D reaction solution, C by the reaction solution for preparing traditional constant temperature exponential amplification techniques (EXPAR) simultaneously
Reaction solution includes inscribe enzyme buffer liquid, template T (0.1 μM), dNTP, target let-7b；D reaction solution includes that polymerization enzyme buffer is molten
Liquid, Vent (exo-) polymerase, restriction endonuclease Nt.BstNBI, SYBR Green I fluorescent dye.C and D is mixed, then 50
DEG C real-time exponential amplification reaction is carried out, with BIO-RAD CFX ConnectTMQuantitative fluorescent PCR monitors amplification curve.
The POI value of 1 two methods of comparative example 3 and comparative example.POI value is smaller, illustrates that amplification efficiency is higher.
The amplification curve of embodiment 3 and comparative example 1 is shown in Fig. 3, and specific POI value is shown in Table 2, it can be seen that the present invention changes
It is 6.50 into type constant temperature exponential amplification POI value, traditional constant temperature exponential amplification curve POI value is 61.5, illustrates that the method for the present invention has
There is better amplification efficiency.
The POI value of the high-order constant temperature exponential amplification (EXPAR) of the present invention of table 2 and traditional constant temperature exponential amplification (TEXPAR) curve
4 specific detection of embodiment
Method:
In order to detect the specificity of the method for the present invention, preparation is a series of to differ 1 or multiple bases with let-7b sequence
Determination concentration let-7a, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i solution.Let-7 family sequence
Column are as shown in table 3.
Prepare A and B reaction solution on ice, A reaction solution includes inscribe enzyme buffer liquid, template T1, template T2, dNTP, above-mentioned
One of target miRNA (one i.e. in let-7a, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i
Kind)；B reaction solution includes polymerization enzyme buffer solution, Vent (exo-) archaeal dna polymerase, restriction endonuclease Nt.BstNBI, SYBR Green
I fluorescent dye.Will A and B mix after be settled to 10 μ L, each group be divided into 0.5 × Nt.BstNBI buffer (25mM tris-HCl,
pH7.90,50mM NaCl,5mM MgCl2, 0.5mM EDTA), template T1 (0.1 μM), template T2 (0.075 μM), dNTP
(1000 μM), target miRNA (100pmol), 1 × ThermoPol polymerize enzyme buffer solution (10mM KCl, 10mM (NH4)2SO4,
20mM Tris-HCl pH 8.8,2mM MgSO4, 0.1%Triton X-100), Vent (exo-) polymerase (0.06U/ μ L),
Nt.BstNBI restriction endonuclease (0.4U/ μ L), 0.4 × SYBR Green I fluorescent dye, DEPC water.Then in 45 DEG C of progress indexes
Amplified reaction monitors each group amplification curve on quantitative PCR apparatus, acquires the POI value of each group POI value and each mispairing situation.
The sequence table of 3 let-7 families of table
Amplification curve POI value of 4 the method for the present invention of table to each member of let-7 family
As a result:
Each group amplification curve is as shown in figure 4, specific POI value is shown in Table 4, it can be seen that the amplification of 100pM let-7b is bent
Line POI value is that other miRNA amplification curve POI values of 4.835,100pM let-7 family are about 10.96~13.25, with blank group
It is close, there is very strong specificity, only with let-7b when illustrating the method for the present invention for detecting target miRNA (such as let-7b)
Interference is also not present to the detection of let-7b in the let-7c of the difference of one base.
The anti-interference evaluation of embodiment 5
Method:
Prepare it is a series of be separately added into chaff interferent let-7a, let-7c, let-7d, let-7e, let-7f, let-7g,
The let-7b solution of let-7i, prepares A and B reaction solution on ice, and A reaction solution includes inscribe enzyme buffer liquid, template T1, template
T2, dNTP, each miRNA；B reaction solution include polymerization enzyme buffer solution, Vent (exo-) polymerase, Nt.BstNBI restriction endonuclease,
SYBR Green I fluorescent dye.A and B two parts solution is mixed, is settled to 10 μ L after A and B is mixed, each group is divided into 0.5
× Nt.BstNBI buffer (25mM tris-HCl, pH 7.90,50mM NaCl, 5mM MgCl2, 0.5mM EDTA), template
T1 (0.1 μM), template T2 (0.075 μM), dNTP (1000 μM), let-7b (10fmol), let-7c~let-7i (10pM), 1
× ThermoPol polymerize enzyme buffer solution (10mM KCl, 10mM (NH4)2SO4,20mM Tris-HCl pH 8.8,2mM
MgSO4, 0.1%Triton X-100), Vent (exo-) polymerase (0.06U/ μ L), restriction endonuclease (0.4U/ μ L), 0.4 × SYBR
Green I fluorescent dye, DEPC water.Then in 45 DEG C of progress exponential amplification reactions, monitoring amplification is bent on quantitative PCR apparatus immediately
Line acquires POI value, and the anti-interference of the modified exponential amplification methods is calculated according to this POI value and standard curve.
Amplification curve POI value when 5 let-7b of table is mixed with other miRNA of let-7 family
As a result:
Each group amplification curve is as shown in figure 5, specific POI value is shown in Table 5, it can be seen that the amplification of 10fmol let-7b
Curve POI value is 7.997, is expanded after mixing respectively with other miRNA of the let-7 family of 10pM, the POI value of amplification curve
It is close with 7.997, illustrate that the method for the present invention can be used for detecting the let-7b containing chaff interferent let-7 other miRNA of family
Solution has stronger anti-interference.Illustrate the strong interference immunity of the method for the present invention detection target miRNA, specificity is high.
The detection of 6 sequence dependent of embodiment
Method:
The let-7b solution to be measured of various concentration (100pM, 1pM, 10fM) is prepared on ice, and prepares identical corresponding concentration
MiR-374a, miR-762 solution to be measured, reaction solution is made into A and B two parts, part A is divided into tri- kinds of A1, A2, A3, A1 packet
Enzyme buffer liquid containing inscribe, template T1, template T2, dNTP, let-7b；A2 include inscribe enzyme buffer liquid, template T3, template T4 (see
Table 6), dNTP, miR-374a；A3 includes inscribe enzyme buffer liquid, template T5, template T6 (being shown in Table 6), dNTP, miR-762.Part B
Comprising polymerizeing enzyme buffer solution, Vent (exo-) polymerase, Nt.BstNBI restriction endonuclease, SYBR Green I fluorescent dye.By A1
And B, A2 and B, A3 and B are mixed respectively, each component concentration be 0.5 × Nt.BstNBI inscribe enzyme buffer liquid (25mM tris-HCl,
PH 7.90,50mM NaCl, 5mM MgCl, 0.5mM EDTA), template T1 (0.1 μM), template T2 (0.075 μM), template T3
(0.1 μM), template T4 (0.075 μM), template T5 (0.1 μM), template T6 (0.075 μM), dNTP (1000 μM), target, 1 ×
ThermoPol polymerize enzyme buffer solution (10mM KCl, 10mM (NH4)2SO4,20mM Tris-HCl pH 8.8,2mM MgSO4,
0.1%Triton X-100), Vent (exo-) polymerase (0.06U/ μ L), restriction endonuclease (0.40U/ μ L), 0.4 × SYBR
Green I fluorescent dye, DEPC water.Reaction solution at 45 DEG C carries out real-time exponential amplification reactions after being sufficiently mixed, and uses BIO-RAD
CFX ConnectTMQuantitative fluorescent PCR monitors amplification curve.
The sequence of 6 miR-374a of table, miR-762, template T3, T4, T5, T6
As a result:
Under the conditions of same concentrations, POI value phase of the method for the present invention to let-7b, miR-762, miR-374a amplification curve
Closely (see Fig. 6 and Fig. 7), no significant difference illustrates that the method for high-order constant temperature exponential amplification detection miRNA of the present invention has low sequence
Dependence, the detection suitable for a variety of miRNA.
The condition optimizing of 7 detection method of embodiment
Method:
A) optimization of reaction temperature
In order to explore influence of the temperature to exponential amplification, under conditions of adding quantitative reagent, mesh under different temperatures is monitored
The quantitative fluorescent PCR curve of miRNA (let-7b) and blank is marked, the POI for calculating corresponding target miRNA and blank curve is poor
Value, the optimum temperature for selecting the maximum corresponding temperature of POI difference to test as exponential amplification.
B) it polymerize the optimization of enzyme dosage
In order to explore influence of Vent (exo-) the archaeal dna polymerase content to exponential amplification, other quantitative reagents is selected to add
Under conditions of adding different content gradient polymeric enzyme, monitor the target miRNA's (let-7b) and blank for corresponding to content polymerase
Quantitative fluorescent PCR curve calculates the POI difference of corresponding target miRNA and blank curve, is guaranteeing the higher base of amplification efficiency
The best polymerase content for selecting polymerase content corresponding to maximum Δ POI to test on plinth as exponential amplification.
C) optimization of nicking inscribe enzyme dosage
In order to explore influence of the nicking restriction endonuclease content to exponential amplification, select other quantitative reagents in addition different content
Under conditions of gradient polymeric enzyme, the target miRNA (let-7b) of corresponding content polymerase and the quantitative fluorescent PCR of blank are monitored
Curve calculates the POI difference of corresponding target miRNA and blank curve, selects most on the basis of guaranteeing that amplification efficiency is higher
The best polymerase content that nicking restriction endonuclease content corresponding to big Δ POI is tested as exponential amplification.
D) optimization of template T2 dosage
In order to explore influence of the chain of exponential amplification template T2 to exponential amplification, the amount of the template of optimum content is selected.Choosing
Select other quantitative reagents monitored on fluorescence quantitative PCR instrument different content template T2 in quantitative objective miRNA (let-7b) and
The amplification curve of blank calculates each a certain amount of template T2 in the difference of quantitative objective miRNA and the POI of corresponding blank amplification curve
Value, difference is bigger to illustrate that background influence is smaller, and T2 corresponding to maximum Δ POI is selected on the basis of guaranteeing that amplification efficiency is higher
Template quantity is optimal Template content.
Concrete operations are as follows:
On ice prepare A and B reaction solution, A reaction solution include inscribe enzyme buffer liquid, template T1, template T2, dNTP,
target(let-7b)；B reaction solution include polymerization enzyme buffer solution, Vent (exo-) polymerase, Nt.BstNBI restriction endonuclease,
SYBR Green I fluorescent dye.When being optimized to temperature, A and B is mixed, then respectively in A 35, B 40, C 45, D
50 DEG C carry out real-time exponential amplification reaction.
When being optimized to polymerization enzyme amount, A and B is mixed, real-time exponential amplification reaction is then carried out at 45 DEG C, DNA is poly-
Synthase content is as follows: A 0.04, B 0.06, C 0.08, D 0.10U/ μ L.
When being optimized to nicking inscribe enzyme amount, A and B is mixed, then real-time exponential amplification reaction is carried out at 45 DEG C, cuts
Quarter restriction endonuclease content is A 0.20, B 0.30, C 0.40, D 0.50U/ μ L.
When being optimized to template T2 amount, A and B is mixed, then carries out real-time exponential amplification reaction, template T2 at 45 DEG C
Content is A 0.005, B 0.075, C 0.100,0.200 μM of D.
This experiment BIO-RAD CFX ConnectTMQuantitative fluorescent PCR monitors amplification curve, calculates each group and corresponds to target
The POI difference of miRNA and blank curve.
As a result:
Testing result is found in 35~50 DEG C, archaeal dna polymerase 0.04~0.10U/ of concentration μ L, nicking inscribe enzyme concentration 0.20
Detection of the present invention to target miRNA is able to achieve under conditions of~0.50U/ μ L, 0.005~0.200 μM of template T2 concentration；But
POI difference according to let-7b and blank is bigger, illustrates that background value is smaller, therefore selects temperature corresponding to maximum Δ POI, interior
The optimum condition that enzyme cutting amount, polymerase content and template T2 content are reacted as this exponential amplification, accordingly, present invention discover that optimal
Reaction temperature be 45 DEG C, the optium concentration of template T2 is 0.075 μM, and the optium concentration of nicking restriction endonuclease is 0.4U/ μ L, DNA
The optium concentration of polymerase is 0.06U/ μ L.
The preparation of 8 standard curve of embodiment
Method: a series of solution to be measured of difference let-7b concentration is prepared on ice；Prepare A and B two parts solution, the portion A
Subpackage inscribe containing Nt.BstNBI enzyme buffer liquid, template T1, template T2, dNTP, let-7b；Part B includes that polymerization enzyme buffer is molten
Liquid, Vent (exo-) polymerase, Nt.BstNBI restriction endonuclease, SYBR Green I fluorescent dye.10 are settled to after A and B is mixed
μ L, each group are divided into 0.5 × Nt.BstNBI buffer (25mM tris-HCl, pH 7.90,50mM NaCl, 5mM MgCl2,
0.5mM EDTA), template T1 (0.1 μM), template T2 (0.075 μM), dNTP (1000 μM), (a series of differences are dense by let-7b
Degree), 1 × ThermoPol polymerize enzyme buffer solution (10mM KCl, 10mM (NH4)2SO4,20mM Tris-HCl pH8.8,2mM
MgSO4, 0.1%Triton X-100), Vent (exo-) polymerase (0.06U/ μ L), restriction endonuclease (0.4U/ μ L), 0.4 × SYBR
Green I fluorescent dye, DEPC water.Then in 45 DEG C of progress exponential amplification reactions, amplification curve is monitored on quantitative PCR apparatus,
POI value is acquired, let-7b concentration mensuration standard curve is obtained.
As a result: amplification curve diagram under different let-7b concentration as shown in figure 8, the standard curve formulated as shown in figure 9, mark
Directrix curve equation is POI=-1.45-0.656lgclet-7b(M), R2=0.984, it is seen that in the miRNA concentration of 100zM-10nM
There is good linear relationship, to the minimum detection limit of miRNA up to 100zM in range.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Zhongshan University
<120>a kind of method that low sequence relies on high-order constant temperature exponential amplification detection microRNA
<130>
<160> 17
<170> PatentIn version 3.5
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Claims (10)
1. the template that a kind of low sequence relies on high-order constant temperature exponential amplification detection microRNA, which is characterized in that the template by
Template T1 and T2 composition, template T1 and T2 are DNA sequence dna；
5 ' the terminal sequences of template T1 and T2 are identical, contain two sections of identical sequence x, contain nicking between two sections of sequence x
The specific site of restriction endonuclease identification, is denoted as x- specific site-x；
3 ' the terminal sequences of template T2 can be with the 5 ' terminal sequence reverse complementals of target microRNA；
3 ' the terminal sequences of template T1 can be located at above-mentioned T2 with the intermediate sequence reverse complemental of template T2, the intermediate sequence of the T2
3 ' terminal sequences and T2 5 ' terminal sequences between；
The intermediate sequence of template T1 can be located at the 3 ' terminal sequence reverse complementals of target microRNA, the intermediate sequence of the T1
Between the 3 ' terminal sequences of above-mentioned T1 and the 5 ' terminal sequences of T1；
In the presence of T1, T2 and target microRNA three are common, two two parts reverse complemental of sequence between three is common to hybridize
Form T-type structure；And under archaeal dna polymerase effect, the 3 ' ends of target microRNA can be expanded with T1 template, be amplified
The sequence with 5 ' terminal sequence reverse complemental of T1, be denoted as x '-specific site-x '；3 ' the ends of T1 can be carried out simultaneously with T2 template
Amplification, the sequence with 5 ' terminal sequence reverse complemental of T2 amplified are denoted as x '-specific site-x '；Above-mentioned x '-specific site-
X ' and x- specific site-x is reverse complementary sequence, and the two combines the double-strand formed that can be cut by nicking restriction endonuclease, in nicking
The site of enzyme cutting cutting is located in x '-specific site-x ' chain.
2. a kind of low sequence according to claim 1 relies on the template of high-order constant temperature exponential amplification detection microRNA,
It is characterized in that, the 3 ' terminal sequences of the template T2 can be with the 5 ' terminal sequence reverse complementals of target microRNA, this section of target
5 ' terminal sequence the length of microRNA account for the 8/21~12/21 of target microRNA total length.
3. a kind of low sequence according to claim 1 relies on the template of high-order constant temperature exponential amplification detection microRNA,
It is characterized in that, the intermediate sequence of the template T1 and the 3 ' terminal sequence reverse complementals of target microRNA, this section of target
3 ' terminal sequence the length of microRNA account for the 8/21~12/21 of target microRNA total length.
4. a kind of low sequence according to claim 1 relies on the template of high-order constant temperature exponential amplification detection microRNA,
It is characterized in that, the length of the sequence x is 14~25nt.
5. a kind of low sequence according to claim 1 relies on the template of high-order constant temperature exponential amplification detection microRNA,
It is characterized in that, the 3 ' terminal sequences of the template T1 can be with the intermediate sequence reverse complemental of template T2, the sequence of this section of reverse complemental
Column length is 8~12bp.
6. a kind of low sequence according to claim 1 relies on the template of high-order constant temperature exponential amplification detection microRNA,
It is characterized in that, the nicking restriction endonuclease is selected from one of Nt.BstNBI, Nt.BsmAI, Nt.AlwI.
7. a kind of low sequence according to claim 1 relies on the template of high-order constant temperature exponential amplification detection microRNA,
It is characterized in that, when object to be measured microRNA is let-7b, the template is TCC ACC GTT TAC TTC TAA TGA
CTC TCC ACC GTT TAC TTC TAA CCA CAA CCC CAA AAA and TCC ACC GTT TAC TTC TAA TGA
CTC TTC CAC CGT TTA CTT CTT TTT TGG GAC CAA CCT ACT ACC TCA TT；
When object to be measured microRNA is miR-374a, the template is TCC ACC GTT TAC TTC TAA TGA
CTC TCC ACC GTT TAC TTC TAA TTA CAA CCC CAA AAA and TCC ACC GTT TAC TTC TAA TGA
CTC TTC CAC CGT TTA CTT CTT TTT TGG GAC ATA CAA TCT GAT AAG TT；
When object to be measured microRNA is miR-762, the template is TCC ACC GTT TAC TTC TAA TGA CTC
TCC ACC GTT TAC TTC TGC TCG GCA CCC CAA AAA and TCC ACC GTT TAC TTC TAA TGA CTC
TTC CAC CGT TTA CTT CTT TTT TGG GAC CCC GGC CCC AGC CCC TT。
8. a kind of method that low sequence relies on high-order constant temperature exponential amplification detection microRNA, which is characterized in that including following step
It is rapid: sample to be tested, the described in any item template T1 for being able to detect target microRNA of claim 1~7 and T2, DNA are gathered
Synthase, nicking restriction endonuclease, dNTP mixing, entire reaction system is reacted under the conditions of 35~50 DEG C, detects amplification curve, according to
Amplification curve and standard curve determine whether contain target microRNA in sample；And target in sample can be further calculated out
The concrete content of microRNA.
9. according to the method described in claim 8, it is characterized in that, archaeal dna polymerase concentration 0.04 in the reaction system~
0.10U/ μ L, nicking inscribe 0.20~0.50U/ of enzyme concentration μ L, 0.005~0.200 μM of template T2 concentration.
10. according to the method described in claim 8, it is characterized in that, also containing DNA polymerase buffer in the reaction system
Liquid, fluorescent dye, nicking inscribe enzyme buffer liquid.
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